NAMPT gene rs2058539 variant is a risk factor for nonalcoholic fatty liver disease

SUMMARY OBJECTIVE: Nonalcoholic fatty liver disease is a chronic liver disease and a growing global epidemic. The aim of this study was to investigate the association between a visfatin gene (NAMPT) variant and nonalcoholic fatty liver disease, owing to the connection between this disease and insulin resistance, obesity, inflammation, and oxidative stress, and the role of visfatin in these metabolic disorders. METHODS: In the present case-control study, we enrolled 312 genetically unrelated individuals, including 154 patients with biopsy-proven nonalcoholic fatty liver disease and 158 controls. The rs2058539 polymorphism of NAMPT gene was genotyped using the PCR-RFLP method. RESULTS: Genotype and allele distributions of NAMPT gene rs2058539 polymorphism conformed to the Hardy-Weinberg equilibrium both in the case and control groups (p>0.05). The distribution of NAMPT rs2058539 genotypes and alleles differed significantly between the cases with nonalcoholic fatty liver disease and controls. The "CC" genotype of the NAMPT rs2058539 compared with "AA" genotype was associated with a 2.5-fold increased risk of nonalcoholic fatty liver disease after adjustment for confounding factors [p=0.034; odds ratio (OR)=2.52, 95% confidence interval (CI)=1.36–4.37]. Moreover, the NAMPT rs2058539 "C" allele was significantly overrepresented in the nonalcoholic fatty liver disease patients than controls (p=0.022; OR=1.77, 95%CI=1.14–2.31). CONCLUSION: Our findings indicated for the first time that the NAMPT rs2058539 "CC" genotype is a marker of increased nonalcoholic fatty liver disease susceptibility; however, it needs to be supported by further investigations in other populations.


INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) as the most common cause of chronic liver disease is a multifactorial disorder in which liver fat exceeds 5% of hepatocytes in the absence of the secondary factors of hepatic fat aggregation or significant alcohol consumption.A growing body of research shows that the prevalence of NAFLD which is currently around 23%-25% of adults in the world will probably increase more owing to the ongoing obesity epidemic.NAFLD patients are more predisposed to have insulin resistance (IR), type 2 diabetes (T2D), and obesity.IR also affects the rate of elevation of serum liver enzymes in NAFLD, and the rate is higher in NAFLD patients with IR than those without IR [1][2][3] .Interestingly, significant associations between insulin receptor (INSR), insulin-like growth factor 1 (IGF1), insulin-like growth factor-binding protein 3 (IGFBP3), and visfatin (NAMPT) gene polymorphisms, and the risk of NAFLD have been discovered [4][5][6][7] .
Nicotinamide phosphoribosyltransferase (NAMPT) or visfatin which is expressed in a variety of tissues including hepatocytes and adipocytes is the product of the NAMPT gene.NAMPT plays a role in the production of nicotinamide adenine dinucleotide.In addition to its aforementioned intracellular enzymatic activity, visfatin also has an extracellular action as a cytokine mainly in inflammation.It appears that visfatin is involved in the development of NAFLD by modulating NAMPT gene and nonalcoholic fatty liver disease risk disorders, including obesity, IR, inflammation, and oxidative stress.Previous reports have indicated positive correlations between circulating visfatin level and IR 8 , T2D 9 , triglycerides 10 , body mass index (BMI) 8,9 , metabolic syndrome 9 , liver enzymes 10 , and NAFLD 11 .Significant associations between NAMPT gene variants and the expression of NAMPT gene 12 and circulating level of visfatin 10 have also been found.Thus, the aim of the current research was to explore the possible contribution of NAMPT gene to NAFLD pathogenesis.The rs2058539 variant of NAMPT gene was selected based on high degree of heterozygosity and its usage in previous research.

Study population
After informed consent, 154 patients with biopsy-proven NAFLD (age range, 31-87 years) and 158 controls (age range, 31-81 years) were enrolled in the present retrospective case-control study.NAFLD patients were enrolled after fatty liver diagnosis which in turn was defined by (a) ultrasonographic confirmation of fatty liver, (b) having high circulating levels of liver enzymes, (c) excluding subjects with other causes of liver disease like Wilson's disease, alpha-1 antitrypsin deficiency, viral hepatitis, and alcohol use more than 70 g/week in women or more than 140 g/week in men, and (d) liver biopsy evidence of NAFLD by an experienced pathologist who examined the liver biopsy samples using the Brunt's criteria.Steatosis and necroinflammation grades were 0-3 and fibrosis stages were 0-4.On the contrary, the control group was recruited from the research staff of the Institute.Those who were free of elevated liver enzymes and viral hepatitis infection (examined by blood tests), had no liver steatosis (examined by abdominal ultrasonography), and were not alcoholic or on regular medications were enrolled as controls.Self-report questionnaires were used for collecting the study population characteristics.This study complied with the principles of the Declaration of Helsinki and was performed according to the Institute's Ethics Committee approval 13,14 .

Genotyping
Blood samples were collected in EDTA vials, and genomic DNA was purified from the white blood cells using phenol chloroform extraction and ethanol precipitation protocol and then stored at -20°C until use.The genotypes of the NAMPT gene rs2058539 variant were determined using PCR-RFLP.Genomic DNA was amplified using the primers: 5'-ATTAACTTTGGTATTCTTGCC-3' and 5'-TAGCCAGTTTTACCTTGAAGAC-3' to detect the genotypes of the NAMPT rs2058539 polymorphism.PCR was carried out with an initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 61°C for 45 s, and extension at 72°C for 45 s.The final extension was at 72°C for 10 min.The PCR products were digested with HphI (Fermentas, Leon-Rot, Germany) in a water bath at 37°C overnight, subjected to agarose gel electrophoresis, and visualized through ethidium bromide (0.5 μg/mL) staining using a UV transilluminator 15 .The "C" allele of the NAMPT rs2058539 had bands of 216 bp and 134 bp, whereas its "A" allele had a band of 350 bp, thus an individual with band(s) at 216 and 134 bp, at 350 bp only, and at 350, 216, and 134 bp was defined as CC homozygotic genotype, AA homozygotic genotype, and AC heterozygotic genotype, respectively.Genotyping of 20% of all the samples was performed twice by different laboratory personnel; the reproducibility was 100%.

Statistical analysis
To perform statistical analyses, the SPSS software package for Windows, version 25.0 (SPSS Inc.Chicago, IL, USA), was used.To examine the normality of distribution of continuous variables, the Kolmogorov-Smirnov goodness-of-fit test was used.The demographic, anthropometric, clinical, and biochemical features of the patients with NAFLD were compared with those found in the controls by Student's unpaired t-test or chi-square (χ 2 ) test as appropriate.Hardy-Weinberg equilibrium (HWE) for the NAMPT gene rs2058539 polymorphism in the control and case groups was also separately analyzed by χ 2 test with 1 degree of freedom, comparing the observed genotype frequencies with those expected.The differences in allele frequencies between the NAFLD and control groups were also assessed using χ 2 test.To appraise the association between the genotype frequencies and NAFLD and to adjust confounding factors, we employed logistic regression analysis.The odds ratios (OR) and their respective 95% confidence intervals (95%CI) were used as the measure of strength for the associations.Differences in biochemical parameters among the different NAMPT genotypes were tested by analysis of variance (ANOVA) and analysis of covariance (ANCOVA) when appropriate.A value of p<0.05 was taken as a statistically significant difference.
Genotype and allele distributions of the NAMPT rs2058539 polymorphism conformed to HWE test both in the case and control populations (p>0.05).This implies that a representative sample population was used in this study.Analysis of this SNP revealed a significant difference between the cases and the controls (Table 2).The "CC" genotype of the NAMPT rs2058539 compared with "AA" genotype was associated with a 2.5-fold increased risk of NAFLD after adjustment for confounding factors (p=0.034;OR=2.52, 95%CI=1.36-4.37).In addition, the NAMPT rs2058539 "C" allele was significantly overrepresented   NAMPT gene and nonalcoholic fatty liver disease risk in the NAFLD patients than the controls (p=0.022;OR=1.77, 95%CI=1.14-2.31).Table 3 shows the association between the rs2058539 variant of NAMPT gene and anthropometric, biochemical, and pathological parameters in 152 patients with biopsy-proven NAFLD; no significant association was found.

DISCUSSION
Genes involved in glucose metabolism, IR, fatty acid metabolism, obesity, oxidative stress, inflammation, and fibrosis development are among the candidate genes for NAFLD.Interestingly, visfatin participates in many of these pathways, hence it is not unreasonable to presume that visfatin and its gene (NAMPT) play a role in NAFLD pathogenesis.The human NAMPT gene that consists of 11 exons is a highly polymorphic gene and has a wide variety of biological functions, so any defects in it may lead to IR, obesity, and inflammation that are implicated in the pathogenesis of NAFLD 16 .In this research, we found that the NAMPT rs2058539 "CC" genotype in comparison to the "AA" genotype increases the risk of NAFLD more than 2.5-fold.Consistently, the NAMPT rs2058539 "C" allele was more frequent in the cases with NAFLD too.Of note, RNA splicing and protein expression may be influenced by alterations in the intronic sequences.Perhaps, the NAMPT rs2058539 variant (located in intron 1) per se not to be functional; instead, it could be in linkage disequilibrium with another functional variant of the NAMPT gene.Consistently, Chang et al. have shown that NAMPT rs2058539 and NAMPT rs61330082 are in a linkage disequilibrium block, and the latter SNP is associated with the transcription rate of NAMPT gene, and serum levels of NAMPT, total cholesterol, triglyceride, and C-reactive protein, as well as cardiovascular diseases 8 .The other possibility is that the rs2058539 "C" allele might be more stable and translates more efficiently into visfatin, which in turn finally increases the risk of NAFLD.Prior investigations have also shown significant associations between NAMPT gene variants and its transcription rate 12 , and circulating levels of visfatin 10 , triglyceride 10 , glucose, and insulin 10 .There have been associations between NAMPT gene polymorphisms and risk of obesity 16 and T2DM 17 too.Visfatin, an insulin-mimetic adipokine, can link IR with obesity.NAMPT is involved in glucose homeostasis and the regulation of glucose-stimulated insulin secretion in pancreatic β cells through NAD biosynthesis, and it induces IR.Interestingly, glucose and insulin regulate the release of visfatin 8,9,18 .Consistently, NAMPT levels were positively associated with triglyceride 10 , BMI 8,9 , IR 8 , T2D 9 , metabolic syndrome 9 , liver enzymes 10 , and NAFLD 11 .There is also more evidence that supports the hypothesis that visfatin plays a role in NAFLD.Visfatin is a proinflammatory cytokine and its level is positively associated with portal inflammation 9 .NAMPT induces the expression of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 via the STAT3 and NF-κB signaling pathways 18 .The NF-κB pathway has a key role in inflammation, and visfatin induces its activation.The injection of visfatin in mice aggravates inflammation, hepatic steatosis, and fibrosis, and increases oxidative stress and plasma levels of liver enzymes 19 .Visfatin also regulates the expression of some important microR-NAs (miRNAs), including miR-146a, miR-155, and miR-181a that participate in inflammation and the activation of the immune system cells.MiRNAs are non-coding and single-stranded RNA molecules containing 22-25 nucleotides, which act in post-transcriptional regulation of gene expression 20,21 .From the above accumulating evidence, it appears plausible to assume that visfatin and its gene, NAMPT, may be involved in the development of NAFLD.
This study had strengths and limitations.First, the sample size of our study was modest due to using liver biopsy.Hence, performing sub-analyses was unreasonable.Second, owing to the fund limitations, we were unable to evaluate serum levels of visfatin.However, the design of this study was good and, more importantly, we employed liver biopsy as the gold standard method for confirming NAFLD diagnosis.Moreover, our report presented some novel and interesting findings.

CONCLUSION
Our findings revealed a role for the NAMPT rs2058539 gene variant in the pathogenesis of NAFLD: the carriers of NAMPT rs2058539 "CC" genotype had a 2.5-fold increased risk for NAFLD.This observation is made for the first time and it supports previous literature; however, further research is needed to confirm it.

Table 1 .
General characteristics of the study groups.

Table 2 .
Distribution of the visfatin gene (NAMPT) rs2058539 variant in the cases with biopsy-proven nonalcoholic fatty liver and the controls a .
a Variables presented as number (%).b Adjusted for age, body mass index (BMI), sex, smoking status, systolic blood pressure (SBP), and diastolic blood pressure (DBP) in genotype-wise comparisons.c Distribution of the NAMPT gene rs2058539 variant in 156 controls and 152 patients.

Table 3 .
Association between NAMPT gene rs2058539 polymorphism and different variables in 152 patients with biopsy-proven nonalcoholic fatty liver.